The long-term objective is to develop diagnostic ultrasound contrast agents (CA) to fulfill the diverse needs of the medical community, and provide useful tools for medical and scientific inquiry. An agent of nano dimensions could fulfill an important diagnostic role by gaining access to areas of pathology from which current micron sized agents are excluded. We published data showing we can produce nano CA (CA-N) with a mean size of 450 nm that have excellent imaging properties. Our most recent studies have produced bubbles of 250 nm. Further investigation is planned. Aim 1: Develop methods to produce 10-500 nm diameter CA by adaptation of the current method for micron- sized agent composed of surfactant-stabilized gas bubbles. ST68-N, upon which preliminary studies have been conducted, is one nano-scale agent composed of Span60 and TweenSO stabilizing an insoluble perfluorocarbon gas bubble. Other members of the Span and Tween family of surfactants are available with differing fatty acid tail groups, and other gases such as sulfur hexafluoride. The hypothesis to be tested is that the wall constituents will dictate the packing density and strength of inter-molecular forces around the gas, hence bubble size and stability. Aim 2: Characterize/optimize in vitro stability and echogenicity. Identify key parameters contributing to the echogenicity of CA-N. Aim 3: Develop methods to incorporate disease- state specific targeting molecules into the bubble wall (tCA), and test them against tissue culture. Solid tumor targeting will be the main focus. Three advantages are gained from targeting: fixation increases the local concentration and allows for greater contrast;in vivo lifetime of tCA is increased by sequestration away from pressure, shear forces, and many of the components of the immune system;and pivotal, it enables distinction between malignant and benign. Aim 4: In vivo characterization including dose response, attenuation, backscatter and Doppler enhancement, study both passive and active targeting of tCA and conduct a nonclinical acute toxicity assessment. We hypothesize that our in vitro cell work will allow us to identify key candidates for CA-N and tCA to be tested in vivo. A large percentage of cancers (approximately 85%) involve solid tumors, and roughly 50% of these lead to metastasis and death. A diagnostic tool that could safely and non-invasively detect tumors at an early stage, before metastasis, would be a great advance in health care.